No effective therapy is currently available to promote recovery from injury or damage of the central nervous system that is associated with an irreversible cell loss. Stem cell transplantation has been proposed for various indications like Parkinson and Huntington disease, amyotrophic lateral sclerosis (ALS), spinal cord injury, syringomyelia, stroke and traumatic brain injury (TBI) (Richardson 2010).
In vitro differentiation of stem cells into desired cell types followed by transplantation into affected region may be of particular value in situations in which a specific cell type is selectively lost and its function is to be restored by replacement of a cell population with similar properties (e.g. ALS, Parkinson) (Burns 2009). In case of TBI, multiple cell types are needed (neuronal, glial, endothelial, vascular etc.) to restore the function of broadly damaged area. Functional neurological improvement accompanied by survival and integration of the implanted cells in injured brain has been reported following transplantation of neural progenitors (fetal cortical and hippocampal cells), marrow stromal cells (MSCs), and even post-mitotic neurons (hNT) derived from human teratocarcinoma cells (Ntera2) after experimental traumatic brain injury (TBI) (Doll 2009).
Stem cells derived from adult tissues such as mesenchymal progenitor cells, fetal progenitors, and post-mitotic cells have a limited potential to differentiate into specific cell types. As TBI is associated with a massive loss of multiple cell types due to primary mechanical tissue disruption, bleeding, and secondary insults such as oedema and increased intracranial pressure leading to cell necrosis, the choice of stem cell population used in replacement therapies after TBI might therefore be critical. Current experimental models of ischemic cerebral injury - stroke, also associated with generalized cell loss, presented us with a gross guideline for transplantation of stem cells following TBI.
Pluripotent murine embryonic stem cells (D3 ES cell line) have been shown to survive and differentiate into neuronal cell types following transplantation into rat brains in an experimental stroke model. Furthermore, GFP-transfected D3 cells have been shown to migrate along the corpus callosum to the ventricular walls and to populate the border zone of the damaged brain tissue on the hemisphere opposite to implantation site, indicating the highly migratory behavior of implanted ES cells (Erdo 2003, Erdo 2007, Hoehn 2002).
Based on these encouraging results, we have implanted undifferentiated D3 cells into the ipsi- or contralateral cortex of rat brains following the induction of a moderate lateral fluid-percussion injury (Riess 2007, Molcanyi 2007).
Timing of transplantation
The timing of stem cell implantation has to be considered as one of the important factors determining stem cell fate in vivo, because traumatic brain injury elicits a dynamic and time-dependent inflammatory response (Knoblach 1998, Stahel 1998, Morganti-Kossmann 2002, Schroeter 2003, Wang 2004). This initial inflammatory burst and the pathological metabolic changes of the acute phase of injury are believed to create an inhospitable micro-environment that might negatively influence implanted ES cells (Okano 2002, Thompson 2005).
In our study, undifferentiated ES cells were implanted 72 hours after fluid percussion injury. This decision was based on reports demonstrating that the initial inflammatory reaction decreases three days after the trauma of nervous tissue (Okano 2002), and is succeeded by the development of astroglial scar within 2 days. The relatively early time point was chosen in order to avoid the peak of any inflammatory reaction and to allow for the migration and differentiation of stem cells that might be obstructed by later formation of astroglial scars (Okano 2002, Thompson 2005).
We evaluated the protective effect of transplantation of undifferentiated, murine embryonic cells on the recovery of motor and cognitive function. We did not observe an improvement of cognitive function. However, a distinct improvement in motor function determined by evaluation of composite neuroscores was observed within 1 week following transplantation of ES cells, indicating a rapid ES cell associated amendment. The trend continued through week 3 and 6 post-transplantation (Fig. 1).
Figure 1 Evaluation of time dependent modification in gross neurological motor function using a composite neuroscore.
White bars: Sham operated animals; grey bars: animals treated with embryonic stem cells following fluid percussion injury; dark grey bars: control animals treated with buffer (PBS). Dots represent individual animal scores, and bars represent median values. * indicates p< 0.05 when injured ES cell transplanted animals are compared to buffer (PBS)-treated injured animals.
Confirming the results obtained by the composite neuroscore, performance in the rotarod test – a functional test developed to analyze sensorimotor coordination - significantly improved post-injury within 1 week following implantation of ES cells, indicating an overall improvement of motor function in ES cell treated animals. The latency remained significantly elevated 3 and 6 weeks post-implantation of ES cells. A modulation of sensorimotor function was not detected when animals were treated with PBS post-injury
These observations are in accordance with previous brain injury studies that also reported recovery of function following cell transplantation. Improved behavioural outcome in sensorimotor and locomotor tests in brain injured animals was demonstrated following transplantation of pre-differentiated ES cells, neuronal precursor cells (C17-2) or minced fetal cortical grafts (E16), respectively (Sinson 1996, Riess 2002, Hoane 2004). However, survival and differentiation of implanted cells or grafts was either not studied or only demonstrated in small numbers, indicating the improvement of the neurological status to be mediated by a small number of surviving and to some extend differentiated cells (Riess 2002, Borlongan 1997, Saporta 1999, Chen 2001, Veizovic 2001).
Stem cell survival and integration
Stem cell fate was examined at 5 days and 7 weeks following implantation. At 5 days post-implantation, labeling of brain sections with anti-GFP antibodies revealed well-defined cell clusters (Fig. 2A) in the peri-injured cortex and subcortical white matter at the contra- or ipsilateral implantation site. Cells, tightly packed into clusters, had a round morphology and a size ranging from 5 to 12 mm. Oct-4 immunolabelling confirmed the presence of undifferentiated ES cells. The outer margin of implanted cell cluster, as well as few isolated cells, distant from the graft, co-localized for a marker nestin, indicating an early differentiation along a neural axis (Fig 2B).
Figure 2. Implantation site, 5 days posttransplantation. A: Section of this region stained with anti-GFP-DAB reveals a brown cluster containing implanted cells.
B: ES cell cluster (appears red - GFP-Cy3) collocalises on its margin for marker nestin (green - nestin-FITC), additionally two remote cells, showing elongated neural-like phenotypes collocalise for this marker of early differentiation.
C: Overview of the ES cell cluster surrounded by macrophages at ipsilateral implantation site - detail of the implantation site - ES cell cluster (appears red - GFP-Cy3) invaded by macrophages (green - ED-1-FITC). Red stem cell remnant is being localised inside of a green circumferential ring – phagocyting macrophage.
At 7 weeks post-implantation, a small number of stem cells was found at the ipsilateral implantation site in only one animal. Migration of the implanted cells was not observed either 5 days or 7 weeks post-implantation.
Effect of brain microenvironment on stem cell fate in vivo
Molcanyi et al. (2007) examined the time-dependent fate of these cells following ipsi- and contralateral implantation into rat brains injured via fluid-percussion injury. Double-staining for GFP and macrophage antigens revealed stem cell clusters embedded in and phagocytosed by infiltrated and activated macrophages, indicating the loss of implanted stem cells was due to an early post-traumatic inflammatory response (Figure 2c). Macrophage infiltration was shown to be less pronounced when stem cells were implanted into completely intact healthy brains. The authors therefore suggested that the massive macrophage infiltration at graft sites might be ascribed to the combined stimulus exhibited by the fluid percussion injury and the substantial conglomeration of stem cells. Previous studies have also shown that the grafting procedure itself, i.e. needle insertion followed by pressure exerted by cell graft infusion, may provoke a notable cellular response in terms of macrophage invasion and microglia activation (Giulian 1989 Persson 1976).
Furthermore, extensive activation of astrocytes throughout the trauma-affected hemisphere has been closely associated with acute inflammatory responses following fluid-percussion injury (Hill 1996). Reactive astroglia expressing GFAP and nestin have also been localized around implantation sites 7-10 days after implantation of a cellular graft (Krum 1999, Krum 2002).
Effect of brain microenvironment on stem cell fate in vitro
The pathophysiological changes associated with TBI seemed to affect the survival, migration and differentiation of transplanted cells and might thereby impair the ability of stem cells to replace lost brain cells and thus diminish trauma induced damage to brain tissue.
Nevertheless, the neurological function, in particular the sensorimotor coordination and motor function was significantly improved up to three weeks after transplantation of stem cells in our study, indicating a protective interaction of the cerebral microenvironment and implanted stem cells.
In vitro studies demonstrated that the mechanism underlying stem cell mediated functional improvement might be partially due to the release of trophic factors by implanted cells (Chen 2002, Bentz 2007). Incubation of D3 stem cells with extract derived from injured rat hemispheres resulted in a rapid time dependent and significant release of BDNF into the medium (Fig. 3) (Bentz 2007).
Figure 3. Time dependent release of growth factor BDNF by treated and untreated stem cells. Treatment groups consisted of cells cultured in serum-free DMEM/Ham’s F12 (untreated; white bars), conditioned with 20% cerebral tissue extract derived from healthy brain hemispheres (grey bars) or with 20% cerebral tissue extract derived from fluid percussion injured hemispheres (black bars). Cells were cultured 3, 5, 7 and 10 days. Values are expressed as Mean ± SEM, p<0.05; *conditioned culture vs. medium only, +time-dependent dynamics, ‡trauma vs. normal brain
It was hypothesized that the supply of trophic support by engrafted cells could be a first impulse in a cascade of events, stimulating the production of neuroprotective substances or even the differentiation of residing progenitor cells leading to a confinement of neuronal damage exerted on the brain, thus attenuating neurological dysfunction and /or even stimulating functional restoration of neuronal circuits (Chen 2002, Chen 2005).
Furthermore, the incubation of stem cells with an extract derived from injured rat hemispheres resulted in a rapid induction of differentiation events in vitro. Stem cells cultivated with brain extract expressed markers of neuronal precursors, such as nestin and MAP2 and developed axonal-like outgrowth (Fig. 4) (Bentz 2010, in press).
Figure 4. Brain extract induced modulation of stem cell morphology at day three following conditioning. Stem cells were conditioned with brain extract derived from traumatized rats. Phase contrast microscopy was carried out at a magnification of 40x.
This data suggests that following TBI, detrimental effects of the cerebral environment are accompanied by protective responses that can potentially induce early differentiation events.
Currently quiet some effort is assigned to the elucidation of the time dependent sequel of injury induced neuronal differentiation, i.e. neurogenesis, and the identification of endogenous factors that might be involved in protective and/or detrimental mechanism following brain injury in vitro and in vivo.
In the present review we have summarized our experience with in-vivo and in-vitro set-ups of cell therapy in environment of cerebral trauma, implementing a standardized model of TBI - fluid percussion injury - reproducing much of the pathophysiology associated with a human closed-head injury (Thompson 2005). Our results clearly demonstrate a detrimental effect of brain microenvironment alterations on stem cell fate. Apropriate time –point of stem cell implantation seemed to be one of the most important aspects determining stem cell fate in vivo, since traumatic brain injury elicited a dynamic and time-dependent inflammatory response.
The influence of brain microenvironment has also been confirmed by novel experiments, utilizing a stimulation of cell cultures by cerebral extract in-vitro. This has lead to selective clonal differentiation of stem cells into neural phenotypes, accompanied by release of neurotrophic factors by stimulated cell populations. Such paracrine effects seemed to play a crucial role in processes leading to neurological improvement, observed after stem cell transplantation in various studies, despite obviously poor long-term survival of implanted graft.
Taking also into consideration an apparent variance of results in injury based cell replacement studies, we believe that a more exhaustive approach towards cell replacement strategies should include a detailed analysis of time dependent pathophysiological modulations following brain injury in varying models.
We hope, this publication will help to acquire more realistic insight into the matter of cell therapy after TBI, for the true danger does not reside in the early initiation of clinical trials, being an issue of medical ethics, but rather in ceasing to perform an accurate scientific search on the front line of this very promising field.
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